Preliminary studies of proteolytic enzymes and steroid receptors in benign and malignant uterine endometrium were performed primarily on cytosols from frozen specimens. Fluorometric assays of cytosols with peptidyl derivatives of aminomethylcoumarin (AMC) revealed high activities (measurable in 10 microliters) of "lysine-specific" and "tyrosine-specific" proteases with different physical-chemical parameters and sensitivities to salts. Gross contamination of many endometrial specimens with blood, and our detection of similar enzymes in cytosols from human leukocytes, however, preclude identification of the source (tissue or blood) of the proteases in some specimens. Endometrial receptors for estrogens (ER) and progestins (PgR) were similar in size and shape to glucocorticoid receptors in cytosols from frozen rat liver. Systematic studies of effects of freezing rat liver, however, revealed decreased receptor size and asymmetry and increased cytosol activity of "lysine-specific" proteases compared with fresh tissue. These problems motivated our current efforts to develop a cell culture system in which to continue the characterization of proteases and steroid receptors and to initiate research on the hormonal control of these enzymes. Transplantation of the selected cell line into nude mice will provide more starting material for enzyme purification. Specific aims include: (1)\determination of whether ER and PgR in cytosols from fresh endometrial specimens and cultured cells are larger than those detected in frozen specimens, as in rat liver; (2)\studies of effects on receptor properties of removal or inactivation of the specific proteases or treatment with purified proteases; (3)\analyses of effects of various steroids in cell culture media on activities of intracellular and secreted proteases; (4)\partial purification of hormoneresponsive enzymes; (5)\search for endogenous protease ihibitors and studies of hormonal effects on inhibitor activity; and (6)\search for and characterization of additional major proteases, e.g., aminopeptidases and collagenases. We will also continue to focus on several other highly active proteases in gynecologic tissues and cells, e.g., those we have detected with tyrosyl and lysyl derivatives of AMC, about which much less is known.